Process for the simultaneous production of d-arabitol, erythritol and glycerol



May 30, 1961 HIROSHI ONISHI 2,986,495

PROCESS FOR THE SIMULTANEOUS PRODUCTION OF D-ARABITOL, ERYTHRITOL AND GLYCEROL Filed April 20. 1959 2 Sheets-Sheet 1 Extraction with 99% ethanol Ethanol solution V (Residue Concentration under reduced pressure Ethanol solution Recrystallzation from Distillation under reduced pressure not ethanol Raw glycerol fraction- D-arabito/ Chromato-pile Vaccurn distillation Erythrito/ fraction 3 Alcohol extrac tion Crystal/ized erytnritol Pure g/yro/ INVENTORI rtttys.

y 30, 1961 HIROSHI ONlSHl 2,986,495

PROCESS FOR THE SIMULTANEOUS PRODUCTION F D-ARABITOL, ERYTHRITOL AND GLYCEROL Filed April 20. 1959 2 Sheets-Sheet 2 Wave length micron Fig. 2

Wave lengfh micron Fig. 3

INVENTOR.

HIROSH! Oil/ISM! BY +fl z United States Patent F PROCESS FOR THE SIMULTALNEOUS PRODUC- TION 0F D-ARABITOL, ERYTHRITOL AND GLYCEROL Hiroshi Onishi, Chiba-ken, Japan, assignor to Noda Industrial and Scientific Research Laboratory, Chiba-ken, Japan, a corporation of Japan Filed Apr. 20, 1959, Ser. No. 807,420

Claims priority, application Japan Feb. 20, 1959 9 Claims. (Cl. 195-37) Type 1: Yeasts being productive for glycerol alone.

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that simultaneous production of glycerol, D-arabitol and erythritol in good yields can be carried out by the fermentation wherein the yeasts stated above are utilized.

The strains which may be most preferably used in the present invention include Pichia miso and Debaryomyces mogii. The former strain was isolated from bean paste (miso) and named as Pichia miso (Mogi: J. Agr. Chem. Soc. Japan 15, 921, 1023, 1221 (1939); ibid. 16, 7, (1940)), and later renamed as Pichia mogii (Ohara and Nonomura: J. Agr. Chem. Soc. Japan 28, 122 (1954)) or as Pichia sake form cc (Kodama: J. Fermentation Technol. 33, 455 (1955) The later strain also was isolated from bean paste (miso) and named as Zygosaccharomyces tikumaensis. Later it was observed from experiment according to Lodder and Kreger van Rij method (1952) that the above strain has a remarkable film-forming property and a poor fermenting activity in an ordinary medium and its cells are round, and therefore this strain was renamed as Debaryomyces mogii. These strains indicated hereinabove would be isolated from bean paste which is subjected to plate culture using koji-agar or 10% NaClkoji-agar, and then purified by single cell isolation.

According to the classification by Lodder and Kroger van Rij (1952), the mycological characteristics of the strains are set forth in Table 1.

TABLE 1 Picnic miso H62 Debaryomyces mogii H71 Vegetative cells (after cultivation in koji extra at C. for 6 days). 7

Growth in koji agar after 2 weeks at 30 C Slide culture on potato agar Ascospore formation Growth in koji extract:

(a) NaOl-iree medium (b) 18% NaGl medium Fermentation of sugars:

(a) Fermented (b) Not fermented Assimilation of sugars:

(a) Assimilated ([2) Not assimilated Assimilation of nitr Growth in ethanol Splitting of arbutin Cells are oval to elongated, (2.5-5) x gill-7) single, in pairs or short ms. The streak culture is white gray colored, dry dull glistening and very wrinkled Primitive pseudomycelium or no pseudomycelium.

Spores are round or short oval 2 x 3 One to 4 spores are found per ascus.

(a) Good growth, dry wrinkled pellicle (b) Good growth, dry wrinkled pellicle (a) Glucose, galactose (b) Maltose, saccharose, lactose (a) Glucose, galactose, saccharose. (b) Maltese, lactose Negativ Good growth Negative, occasionally weakly positive.

Cells are round to short oval, 4-6u,

single, in pairs or short chains.

The streak culture is white gray colored, dry dull glistening and wrinkled.

No pseudomycelium.

Spores are round, 2-3 One to 3 spores are found per ascus.

(a) Good growth, dry wrinkled pellicle.

((2) Good growth, dry wrinkled pellicle.

(a) Glucose, galactose. (b) Maltose, saccharose, lactose.

(a) Glucose, galactose, saccharose. (b) Maltese, lactose.

Negative.

Good growth.

Negative.

Among these types of yeasts, only those of Type 4 involving Zygosaccharomyces genus and Saccharomyces rouxii (osmophilic Saccharomyces) are well known as producing the polyhydric alcohols other than glycerol. (Cf. Appl. Microbiol. 6, 349 (1958); Can. J. Microbiol. 2, 72 (1956); U.S.P. 2,793,981 (1957).) Now it has been discovered that the film-forming yeasts of Pichia genus or Debaryomyces genus belong to Type 6, and they can produce an exceptionally large amount of polyhydric alcohols by fermentation.

These yeasts of Pichia and Debaryomyces genera are oxidizing type in the metabolic system, whereas those of Zygosaccharomyces and Saccharomyces genera are fermenting type. Heretofore it was considered that the yeasts which may be used in the present invention are commercially useless, and in some cases e.g. brewage of soy sauce, bean paste or salted pickles, they are regarded as noxious. Accordingly, it is surprising that we have now found Both of the yeasts indicated in the above table are highly sugarand salt-tolerable, and they can utilize completely a concentrated solution of 30% glucose.

The yeasts of the aforesaid genera are tested for the production of polyhydric alcohols by carrying out the following procedures. That is to say, freshly cultured strains are inoculated into 500 ml. shaking flasks containing 80 ml. of an aqueous medium having a pH of 4.8 and containing:

, Percent Glucose 30 KH PO 0.1 MgSO -7H O Bo CaCl -2H O 0.01 NaOl 0.01 Casamino acid 0.4 Yeast extract 0.1

paper chromatography to determine qualitatively polyhydric alcohols. In other words, the polyhydric alcohols in the fermentation broth are developed using a mixed solvent (n-propanolzethyl acetate:water=7:1:2) (cf. Anal.

The D-arabitol thus obtained is in the form of prismatic crystals melting at 102 C. and has a little sweet taste. Its empirical formula is C H O and Chem. 27, 1400 (1955)) and then detected according to 5 Yodas method (of. J. Chem. Soc. Japan 73, 18 (1952)). (C=9.78, saturated boraX solutlon). As seen from the These analytical results are shown in the following table. data of infra-red spectrum (Fig. 2), the D-arabitol crys- TABLE 2 Polyhy- Yields of Polyhydric alcohols produced Glucose dricalcohols polyhydric Strain consumed, produced alcohols percent (as glyc (as glycerol), crab, percent percen D-arabitol Erythrltol Glycerol Pivhia miw 5 28.6 12. 24 42. 4 t "W moaii 7 29.5 11.44 38.1 mlmdshurica (Control) 1e, 3 1. 90 11. 7

1 Yields of polyhydric alcohols as glycerol are calculated based on the amounts of glucose consumed.

D-arabitol are respectively determined by subjecting the I broth to paper chromatography and then eluting each of the developed spots with water.

V alcohols.

tal obtained in the present invention is identical with anthentic D-arabitol. The erythritol crystal obtained in the present invention is inthe form of tetragonal prisms and has a melting point of 121 C. and a sweet taste. Empirical formula C H O The infra-red spectrum (Fig. 3) shows that the erythritol obtained by the present invention is identical with authentic erythritol.

The following tests wherein a strain Pichia miso H52 is employed are carried out in order to determine various cultivation conditions for the production of polyhydric At the same time, these tests will serve to understand the characteristics and features of the present invention more clearly.

Glucose concentration TABLE 3 As understood from the following Table 4, the glucose Glucose consumed percent 29.47 concentration of from 30% to 40% is most preferred to Polyhydric alcohols produced (calculated as the production of polyhydric alcohols. When the gluglycerol) percent 11.5 cose concentration is about 10%, the production of poly- Yield of polyhydric alcohols from glucose conhydric alcohols is considerably low. As to the types of sumed "percent" 39 .0 polyhydric alcohols, D-arabitol is well produced inde- D-arabitol produced do 8.5 pendently of the glucose concentration, whereas erythri- Yield of D-arabitol from glucose consumed tol can be produced only at the glucose concentration percent 28.8 ranging from 30% to 40% and glycerol is scarcely pro- Erythritol produced do 0.6 40 duced at the glucose concentration of 10%.

TABLE4 Yield of Polyhydrlc polyhydric Glucose concen- Glucose alcohols alcohols tratlonlnculconsumed, produced from Polyhydrlc alcohols produced ture medium, percent (as glycglucose percent erol), consumed percent (as glycr p r ent D-arabitol Erythritol Glycerol 9. s2 1. 84 1s. 7 i 19.19 6.13 31.9 27. 11. 91 4a. 1 23.15 9. so 42. 3 15. 52 5. 20 33. 5

Yield of erythritol from glucose consumed percent-.. 2.0 Glycerol produced do 5.9 Yield of glycerol from glucose consumed percent" 20.0 Riboflavin produced ;tg./ml 2.1

Eflect of added nitrogen source As to the nitrogen source of culture medium, both of ammonia N and amino N are suitable for the satisfactory production of polyhydric alcohols. Now it should be noted that in a case wherein inorganic nitrogen sources are utilized, the pH of culture medium rapidly decreases to 2.0 or less than at the initial stage of the fermentation and as its result the production of polyhydric alcohols is reduced. Therefore, if an inorganic compound is used as a nitrogen source, a butter solution (e.g. K-citrate and citric acid butter) must be added to the culture medium to prevent the depression of a pH of said medium during 76 the. fermentat on.

TABLE Polyhydrlc Yield of Polyhydrlc alcohols Glucose alcohols polyhydrio Nitrogen Source consumed, produced alcohols percent (as glyc- (as glycerol), erol), D-arabitol Erythrltol Glycerol perccnt percent Meat extract, 0.4% 11.88 4. 64 39.1 :1: Corn steep liquor, 0.4% 20. 24 8. 78 43. 3 Polypeptone, 0.4% 18. 24 9. 98 54. 7 1 Yeast extract, 0.4% 28.80 11.19 38. 8 Ammonium lactate, 0 2% 28. 85 12. 09 41. 9 Urea, 0.05% 23. 98 10.15 42.3 Ammomum sulfate, 0 Not added with buffer 12. 32 2.07 16. 8

sulfate, 0.2% Added 2 W1 u er 7. 54 9. 46 34. 3 Ammonium chloride, 0.2% Not added with buffer 11.69 1.81 15.4 Ammonium chloride, 0.2% Added with bufier 23. 32 7. 79 33.4

Efiect of the type of sugar As shown in the following Table 6, polyhydric alcohols can be remarkably produced by the fermentation of glucose, fructose or mannose, but scarcely produced by that of galactose, maltose or saccharose.

Fig. 2 is a curve of the infra-red spectrum of D-arabitol produced by a method according to the present invention. Fig. 3 is a curve of the infra-red spectrum of erythritol produced by a method according to the present invention. While the present invention has, for convenience, been TABLE 6 Polyhydrlc Yield of Polyhydrlc alcohols produced Sugar alcohols polyhydrlc Culture medium containing consumed, produced alcohols percent (as glyc- (as glycerol), eroll, D-arabltol Erythrltol Glycerol percent percent 30% Glucose 28.6 12. 24 42. 4 +++-1- 30% Fructose 26.4 8. 22 31.1 30% Mannose 19. 1 6. 49 33.9 30% Galactose". 11.3 0.12 1. 1 30% Maltese 0.3 0.04 18. 3 30% Sacoharose 10. 1 0. 14 1. 3

Efiect of aeratzon 0 described 1n connection with a strain Pichia miso, also When 500 ml. shaking flask containing 30 to 80 ml. of the culture medium is subjected to shaking culture under the conditions indicated hereinabove, the formation of polyhydric alcohols in good yields is obtained. In this case, Kd value measured by the sodium sulfite titration (cf. J. Agr. Chem. Soc. Japan 27, 704 (1953)) is 2.5 g. mol. of O /atm. min. cc. or higher. Ho ever, when the shaking flask containing 120 ml. or more than of the culture medium is cultivated in the same conditions as above, the formation of polyhydric alcohols is inferior to the above case. (In this case Kd value is less than 1.7 X10 other osmophilic, film-forming yeasts may be used for the simultaneous production of polyhydric alcohols. Since many difierent embodiments of the invention may be made without departing from the spirit and scope thereof, it is to be understood that we do not limit ourselves except as set forth in the following claims.

What I claim is:

1. A method for the production of D-arabitol, erythritol and glycerol, which comprises aerobically culturing a microorganism selected from the group consisting of Pichia miso and Debaryomyces mogii at a temperature Effect of temperature The present yeast can be grown between l5-40 C. in the above medium. Optimum temperature for the production of polyalcohol is -35 C.

Referring to the drawings:

Fig. 1 is a flow sheet of a suitable process for the isolation of D-arabitol, erythritol and glycerol from the fermentation broth which has been fermented according to the present invention.

of 15 to C. in a medium containing 30 to by weight of fermentable sugars selected from the group consisting of glucose, fructose and mannose, and 0.05 to 0.4% by weight of a nitrogen source selected from the group consisting of an organic nitrogen source and a mixture of an inorganic nitrogen source with a buffer, whereby D-arabitol, erythritol and glycerol are simultaneously produced.

2. A method according to claim 1, wherein the organic "7 nitrogen source is selected from the group consisting of casein hydrolyzate, amino acid, meat extract, corn steep liquor, polypeptone, yeast extract, ammonium lactate and urea.

3. A method according to claim 1, wherein the inorganic nitrogen source is selected from the group consisting of ammonium sulfate and ammonium chloride, and the buffer is potassium citrate-citric acid buffer.

4. A method according to claim 1, wherein the temperature is 30 to 35 C.

5. A method according to claim 1, wherein the culture medium also contains minor amounts of potassium phosphate, magnesium sulfate, calcium chloride, sodium chloride and vitamins. Y

6. A method according to claim 1, wherein the aerobic culturing is carried out by aeration.

, 7. A method according to claim 1, wherein the aerobic References Cited in the file of this patent UNITED STATES PATENTS Haehn Feb. 13, 1940 Spencer et a1. May 28, 1957 

1. A METHOD FOR THE PRODUCTION OF D-ARABITOL, ERYTHRITOL AND GLYCEROL, WHICH COMPRISES AEROBICALLY CULTURING A MICROORGANISM SELECTED FROM THE GROUP CONSISTING OF PICHIA MISO AND DEBARYOMYCES MOGII AT A TEMPERATURE OF 15* TO 40*C. IN A MEDIUM CONTAINING 30 TO 45% BY WEIGHT OF FERMENTABLE SUGARS SELECTED FROM THE GROUP CONSISTING OF GLUCOSE, FRUCTOSE AND MANNOSE, AND 0.05 TO 0.4% BY WEIGHT OF A NITROGEN SOURCE SELECTED FROM THE GROUP CONSISTING OF AN ORGANIC NITROGEN SOURCE AND A MIXTURE OF AN INORGANIC NITROGEN SOURCE WITH A BUFFER, WHEREBY D-ARABITOL, ERTHRITOL AND GLYCEROL ARE SIMULTANEOUSLY PRODUCED. 